Unprecedented NO production by nitrate-ammonifying with distinctive NO isotopocule signatures.

Dissimilatory nitrate reduction to ammonium (DNRA), driven by nitrate-ammonifying bacteria, is an increasingly appreciated nitrogen-cycling pathway in terrestrial ecosystems. This process reportedly generates nitrous oxide (NO), a strong greenhouse gas with ozone-depleting effects. However, it remains poorly understood how NO is produced by environmental nitrate-ammonifiers and how to identify DNRA-derived NO. In this study, we characterize two novel enzymatic pathways responsible for NO production in strains, which are predominant nitrate-ammonifying bacteria in paddy soils. The first pathway involves a membrane-bound nitrate reductase (Nar) and a hybrid cluster protein complex (Hcp-Hcr) that catalyzes the conversion of NO to NO and subsequently to NO. The second pathway is observed in Nar-deficient bacteria, where the nitrite reductase (NrfA) generates NO, which is then reduced to NO by Hcp-Hcr. These enzyme combinations are prevalent across the domain Bacteria. Moreover, we observe distinctive isotopocule signatures of DNRA-derived NO from other established NO production pathways, especially through the highest N-site preference (SP) values (43.0‰-49.9‰) reported so far, indicating a robust means for NO source partitioning. Our findings demonstrate two novel NO production pathways in DNRA that can be isotopically distinguished from other pathways.IMPORTANCEStimulation of DNRA is a promising strategy to improve fertilizer efficiency and reduce NO emission in agriculture soils. This process converts water-leachable NO and NO into soil-adsorbable NH, thereby alleviating nitrogen loss and NO emission resulting from denitrification. However, several studies have noted that DNRA can also be a source of NO, contributing to global warming. This contribution is often masked by other NO generation processes, leading to a limited understanding of DNRA as an NO source. Our study reveals two widespread yet overlooked NO production pathways in , the predominant DNRA bacteria in paddy soils, along with their distinctive isotopocule signatures. These findings offer novel insights into the role of the DNRA bacteria in NO production and underscore the significance of NO isotopocule signatures in microbial NO source tracking.